Hyperhomocyst(e)ineimia is a risk factor for the development of atherothrombosis, and is believed to exert its adverse vascular effects, in part, through a mechanism involving oxidant injury. Homocysteine is readily oxidized when added to plasma by auto-oxidative mechanisms that lead to the formation of homo- and heterodisulfides, homocysteine thiolactone, superoxide anion, and hydrogen peroxide. The central hypothesis of this competing renewal proposal is that pathophysiologically relevant concentrations of homocyst(e)ine impart an oxidant stress to the vascular microenvironment that leads to a state of endothelial dysfunction manifest by reduced production of bioactive nitric oxide. This hypothesis will be addressed through three specific aims. Firstly, we will define the molecular mechanism(s) by which pathophysiologically relevant concentrations of homocyst(e)ine induce endothelial oxidant stress. We will do so by measuring the effect of homocyst(e)ine on the intracellular production and sources of reactive oxygen species and the concomitant decrease in intracellular redox state in cultured endothelial cells. We will also focus, in particular, on a critical enzymatic determinant of cellular redox state, cellular glutathione peroxidase. The expression of this selenocysteine-containing antioxidant enzyme is uniquely suppressed by homocyst(e)ine, and we will study the molecular mechanisms by which its expression is regulated by homocyst(e)ine. Secondly, we will examine the consequences of increased homocyst(e)ine and reactive oxygen species concentrations on endothelial production of bioactive nitric oxide, focusing specifically on mechanisms of decreased synthesis by concomitant decreases in endothelial cofactors essential for nitric oxide synthase activity. We will also attempt to reverse the effects of homocysteine on endothelial nitric oxide production and bloactivity by restoring depleted cofactors, suppressing oxidant stress, or both. Lastly, we will apply these in vitro observations to in vivo models of mice in which the cystathionine-b-synthase or glutathione peroxidase-1 genes have been inactivated by targeted disruption in an effort to define the role of lipid peroxides and derivative reactive oxygen species on endothelial dysfunction and decreased production of bioactive nitric oxide. We will attempt to reverse these adverse oxidant effects of hyperhomocyst(e)inemia by the use of selected antioxidants and by crossing the cystathionine-b-synthase-deficient mice with mice that overexpress cellular glutathione peroxidase. With these studies, we hope to gain insight into the molecular mechanisms of homocyst(e)ine-induced endothelial dysfunction and potential therapies for this common metabolic abnormality.